The goal of this project is to define the molecular mechanism of calcium transport across the intestine, and to provide a molecular understanding of the effects of vitamin D and parathyroid hormone in the control of calcium absorption and secretion. The methods to be used involve radioactive tracer, electrophysiological, and fluorescence techniques to measure fluxes across the intact intestinal epithelium, and across isolated brush border and baso-lateral plasma membranes. The purified plasma membranes will be obtained by a preparative scale isolation procedure employing density gradient centrifugation in high capacity zonal rotors. The membranes exist as osmotically active sealed vesicles and so it is possible to measure the calcium fluxes across these isolated plasma membrane fractions. The energetics of calcium transport will be studied by determining the relationship between demonstrated calcium stimulated ATPase activities and ATP-driven calcium transport. Identification of a calcium transporting enzyme in epithelial cell plasma membranes will further permit us to study possible molecular responses to Vitamin D and parathyroid hormone. In order to arrive at an integrated understanding of the mechanisms of calcium transport and its control, we will reconcile the results obtained with the isolated plasma membranes with measurements of calcium transport in the intact epithelium. Long term plans include the isolation, purification and characterization of the enzyme responsible for calcium transport, and the reconstitution of calcium transport in phospholipid liposomes.